This application claims priority from Korean Patent Application No. 10-2004-0013779, filed on Feb. 28, 2004, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to a micro polymerase chain reaction (PCR) device, a method for amplifying nucleic acids using the micro PCR device, and a method for measuring the concentration of PCR products using the micro PCR device.
2. Description of the Related Art
A traditional PCR provides the qualitative results of amplified DNAs by an electrophoresis at the end-point of PCR, but has many problems such as inaccuracy of the quantitative detection of DNAs. Therefore, a Real-Time PCR has been developed to allow for the quantitative assay of amplified DNAs by detecting the intensity of a fluorescent signal, which is in proportional to the concentration of the amplified DNAs, using an optical detection system.
A quantitative assay of DNAs is essential for studying disease treatments and DNA expression. For example, in order to ensure a successful medicinal therapy for patients infected with hepatitis B virus (HBV), the drug resistance to HBV must be tested by periodically detecting the concentration of HBV in a blood stream using a Real-Time PCR.
A conventional Real-Time PCR requires many optical devices such as a laser source, a micromirror, a microscope, and a filter, and an expensive fluorescent dye. In addition, because a conventional Real-Time PCR chip is based on the principle of detecting a fluorescent signal, there are many disadvantages in terms of miniaturization (chip formation) and economical efficiency.
In order to solve this problem, an effort was made to electrically detect DNAs using capillary electrophoresis (CE). This method allows for a qualitative assay, but has many problems for a quantitative assay. In addition, the transfer of PCR products to a CE detection system using a micro-channel after the completion of PCR is a laborious process and a high voltage is required. Therefore, requirements of economical efficiency and miniaturization are not satisfied.
U.S. patent application Publication No. 2002/0072054A1, filed by Miles et al., is based on the concept that as the concentration of DNAs increases during PCR, impedance decreases and conductivity increases. However, this application is concerned with a PCR chip for end-point detection and has no relevance to Real-Time PCR. In addition, there is a problem in that an ionically labeled probe must be used to detect amplified PCR products.
Meanwhile, according to conventional methods for detecting PCR products using an electrical or chemical signal as described above, measurement sensitivity and reproducibility are low, which makes it difficult to obtain reliable results. This is because components of a PCR mixture, such as proteins, ions, and stabilizers, are nonspecifically adsorbed to surfaces of electrodes detecting an electrical signal.
The present invention has been made in view of these problems. According to the present invention, one primer of a primer set is immobilized on surfaces of electrodes disposed in a PCR chamber, and the other primer labeled with nanoparticles is added to a PCR mixture. PCR is performed on the surfaces of the electrodes using the primer set and PCR products can be detected in real-time. Therefore, high detection sensitivity and reproducibility can be ensured. Furthermore, since various electrochemical detection methods can be used in detecting the PCR products, accuracy of the detection result can be increased.
Such performance enhancements of the present invention can be elucidated as follows. When self-assembled monolayers are formed by immobilizing primers on the surfaces of electrodes, nonspecific adsorption of other nucleic acids or proteins on the surfaces of the electrodes is almost completely prevented. Furthermore, since electrochemical detection is carried out for components immobilized on the surfaces of the electrodes, not the PCR mixture, detection sensitivity is enhanced. In addition, since the PCR products are immobilized on the surfaces of the electrodes, factors inhibiting the electrochemical detection can be removed by washing the electrodes, thereby enhancing reproducibility.